Lovastatin (mevinolin; monacolin K; .beta.,.delta.-dihydroxy-7-[1,2,6,7,8,8a-hexahydro-2,6-dimethyl-8-(2-methy l-butyryloxy)-naphthalen-1-yl]-heptanoic acid .delta.-lactone) is one of the most important known cholesterol lowering agents. Lovastatin, as used herein, includes both the lactone and free hydroxy acid forms.
Its open hydroxy acid form is a potent inhibitor of the 3-hydroxy-3-methyl-glutarylcoenzyme A reductase enzyme, which catalyzes the formation of mevalonic acid, an early intermediate of cholesterol biosynthesis. Lovastatin is specifically advantageous because, as a result of its application, biosynthetic intermediates with a toxic steroid skeleton, formed at a later stage of biosynthesis fail to accumulate. Lovastatin increases the number of LDL-receptors at the surface of the cell membrane which remove the LDL cholesterol circulating in the blood, thereby inducing the lowering of blood plasma cholesterol level.
Commonly, the active ingredient is produced via fermentation. GB 2,046,737 discloses that the active ingredient can be produced by some strains belonging to the Monascus genus, e.g., by M. ruber 1005 cultivated between 7 and 40.degree. C. As a culture medium, an aqueous solution of glucose, peptone, corn steep liquor and ammonium chloride was used. The fermentation was carried out for 10 days in aerobic conditions, and 87 mg Lovastatin was obtained from the filtrate of 5 liters of broth.
U.S. Pat. No. 4,294,926 discloses the biosynthesis of lovastatin preferably by the application of microorganisms under the deposited numbers ATCC 20541 or 20542 belonging to the Aspergillus terreus species on a culture medium containing carbohydrates, e.g., glucose, fructose, maltose, as carbon source; nitrogen sources, e.g., yeast, hydrolyzed yeast, hydrolyzed casein, corn steep liquor; and mineral salts, e.g., calcium carbonate, magnesium sulfate, cobalt, iron, and manganese salts at a temperature of 20-37.degree. C. Similar procedures are described in U.S. Pat. Nos. 4,420,491, 4,342,767, 4,319,039 and 4,294,846, where the fermentations are carried out for 3-5 days on media containing 1-6% carbohydrates and 0.2-6% nitrogen sources.
German Patent No. 4,402,591 discloses biosynthesis of Lovastatin by microorganisms belonging to the Pleurotus genus, e.g., P. ostreatus, P. sapidus and P. saca, at 25-35.degree. C. during 7-14 days cultivation time on surface or submerge cultures.
Canadian Patent No. 2,129,416 discloses the preparation of lovastatin, or in a particular case, mevastatin, with a microorganism belonging to the Coniothyrium genus, e.g., under the deposited number Coniothyrium fuckelii ATCC 74227 on a culture medium containing 3-15% glucose, 0.5-4% peptone, 0.5-5% amylase, 0.2-1% ammonium sulphate, 0.01-0.1% magnesium sulphate, 0.05-0.2% antifoaming agent, 0.2-1.5% L-isoleucine, 0.2-1.5% L-aspartic acid in the pH range of 5-6. According to the examples, the active ingredient concentration of the broth was within 19-430 mg/liter.
Hungarian Patent No. HU 208,997 discloses the application of the holotype strain Aspergillus obscurus numbered as MV-1, deposited under the number NCAIM(P)F 001189. The fermentation is preferably carried out on a medium containing yeast extract and/or peptone and/or casein as nitrogen source(s) and glucose and/or maltose or sucrose as carbon source(s). The activity of the broth at the end of the laboratory scale cultivation is between 400-850 mg/liter.
The foregoing discussion establishes that the development work in the biosynthesis of lovastatin focused on discovery of new lovastatin-producing microorganisms rather than on the development of the fermentation procedure itself. Several references disclose that fermentations can be carried out on conventional and known media with the application of both surface and solid state cultivations. Batch-like procedures were applied where the behaviors of the procedures depended on the initial conditions. However, technical limitations, e.g., maintaining the most convenient level of ingredients, optimal dissolved oxygen supply and pH, etc., made it difficult to implement continuous corrective actions to ensure more favorable conditions. A given microorganism during the main fermentation stage, depending on its metabolism, requires different conditions/composition of media in order to obtain optimal growth and production of active ingredient. The present inventors concluded from their experiments that in the seed culture and at the beginning of the main fermentation, the quantity of active biomass is very small and variable. Thus, the yield of the fermentations are relatively low and variable. Yields reached at the end of the fermentations, which depended of course on the strain, did not exceed a lovastatin concentration of 850 mg/liter. The present inventors performed a detailed analysis of the whole fermentation procedure from the seed culture stage throughout the end of the fermentation. It was found that in the seed culture preparation stage, both in the case of the known media and execution processes, the quantity of the biomass is too low. Therefore, during the main fermentation, the metabolism of the microorganism and the morphology of the culture are not adequate.